The devices and methods of the prior art for performing precise immunological diagnoses generally use devices having most often already been the object of an immunological preparation (by immobilization of a first antibody). Thus the wells of the plates used in the ELISA type processes comprise in general a coating comprising first antibodies to capture an antigen, which will then be recognized by a second antibody, the whole being then revealed by a third anti-immunoglobulin antibody. The good practice of this process requires at least six washings and three hours of incubation.
Rapid tests have appeared during the 1980s, that use microballs previously clad with a first antibody and which migrate on a nylon surface to come to rest ultimately on a first line (presence of antigens) of second antibodies, for stopping in all cases on a second line of third antibodies. The whole takes place in several minutes, using a minimum of 7 nanograms of antigen per milliliter of specimen. This process does not permit detecting serum antibodies.
The object of the present invention is no longer an electrophoresis which is characterized by a more or less prolonged migration between two electrical poles (one at the beginning and the other at the end), as a function of molecular weight of different proteins. There exist more or less sophisticated gel techniques conducted between two electrodes and which permit separating a group of proteins in different bands according to their molecular weight.
The present invention has for its object to overcome the insufficiency of sensitivity of the known methods, in particular to provide a process permitting obtaining a qualitative or quantitative signal of the presence, at infinitesimal concentrations, of a substance in fluids of a specimen, even if it is not constituted by a nucleic acid. The so-called PCR (polymerase chain reaction) technique detects very small quantities of fragments of chromosomic material (RNA or DNA). It is not capable of measuring proteins such as new generations of tumoral markers or cytokenes circulating at quantities that can be less than a picogram per milliliter (a billionth of a gram). The immunological techniques, even the most sensitive such as radioimmunoanalysis, do not permit it.
The process according to the invention is applicable to the detection of any analyte that may be contained or adapted to be included in a fluid or a liquid which can react with a specific ligand to form a complex, the analyte or the ligand or both being carriers of electronic charges which are neutralized, or even reverse in the complex when the latter is formed. It is characterized in that:
the fluid or liquid is caused to migrate, after placing it in contact with the specific ligand, in an electrically inert porous material interposed between opposite surfaces of two electrically charged electrodes of the same polarity, and
there are detected in the liquid that has migrated into the porous material between the two electrodes, those components, analyte, ligand or complex, which were not captured by the electrodes.
Needless to say, the total volume of fluid or liquid used, which is that contained in the dissolved condition of one of said components or both, particularly as a result of their possible mixing, must be sufficient to permit its diffusion in the porous material to the place where the detection is carried out.
According to the nature of the constituent detected, or not detected, therefore depends the conclusion that can be drawn as to concentration. In a preferred embodiment of the method according to the invention, one of the constituents, for example the ligand, is marked. It is for example an antibody which carries free NH2 groups, therefore positive charges (the analyte being thus an antigen), and if the electrodes are negatively charged, the detection of the marker in the fluid or liquid having migrated into the porous material between the electrodes will show that the antibody is engaged in a complex, hence that the antigen was present in the original liquid or fluid. Thus following the antigen-antibody reaction, the NH2 groups of the antibodies are masked in the formed complex, the positive charge of the antibody being thus neutralized, the antigen-antibody complex being again, as the case may be, able to have a negative polarity. On the contrary, the absence of detection will translate into the absence of the antigen in the fluid or liquid studied. Thus the marked antibodies which have not encountered xe2x80x9cpartnersxe2x80x9d are thus captured and blocked by the negatively charged electrodes.
Other examples show in what follows this description, important possibilities offered by the invention. But for good understanding of the invention, it may be useful to define the expressions xe2x80x9canalytexe2x80x9d and xe2x80x9cligandxe2x80x9d, as used in the present description. Thus, the application of the invention is not limited to the detection of analytes constituted only by antigens or antibodies, even if it is in this connection that the invention will be most often used.
By analyte, should be understood any molecule or entity which it is desired to detect, no matter what its nature: haptene, protein, antigen, antibody, nucleic acid fragments, substance, even a group of molecules having common characteristics, this analyte being adapted to give rise to a specific reaction with a ligand to form a complex adapted to take part in a reaction seeking to detect in a selective manner the presence or absence of the analyte in a fluid or a liquid, for example a fluid of biological origin.
In the case of an analyte constituted by an antigen, it will be appreciated that the antibody, which thus plays the role of the ligand, is the carrier of NH2 sites which give it a positive polarity, giving rise to the possibility for this antibody to be captured by an electrode having negative charges, whether the latter is of electrostatic nature or the result of placing the electrode under voltage in the suitable direction. Naturally, the converse can also take place. It can happen, for example, that in other types of analyte-ligand couples, the polar group will be formed from a negatively charged group, for example a COOH group, which will be neutralized in the analyte-ligand complex that is formed.
Accordingly, the ligand can as needed be itself modified by a group giving it its properties, for example the required electrical polarity for its use in the method of the invention. This could, by way of example, be the case when the analyte is constituted by a DNA fragment, whose presence can be searched for in a liquid after it has itself been used in a complementary RNA detection test. In such a case, the DNA fragment will be for example rendered the carrier of a biotin group, the ligand thus acceptable to be used-being adapted to be constituted by avidine, carrying itself, as the case may be, a marker, for example colorimetric, fluorescent, chemiluminescent, radioactive or the like and, if there was further need, a supplemental group giving it an electrostatic charge, which could thus be neutralized during the production of the analyte-ligand complex whilst susceptible to being formed, via the biotin-avidine reaction. Or else the analyte could be constituted by a DNA fraction and the ligand by a DNA probe itself marked by an enzyme, particularly peroxidase, by means of spacer arms produced by means of heterobifunctional systems, for example of the (DNA (FMCC)-peroxidase (SPDP) type.
Preferably, the analyte is in solution in a liquid or a fluid. But for example when the analyte consists of an antigen, it can also be contained for example in a biopsy or mucous fragment. But it then should be extracted by, or carried in, the liquid which will then migrate in the porous material between the electrodes.
As to the xe2x80x9cporousxe2x80x9d material, it must necessarily be meant by this expression any material permitting to propagation or diffusion of a liquid through its mass, when it has been moistened with this liquid. It could be constituted of any open pore material, fibrous material or which can behave as an absorbent, etc . . .
As to the electrodes, use can be made of any material adapted to be electrically polarized or electrostatically charged. Numerous materials in the form of thin sheets are available commercially: see for example the chapter beginning at page 53 of the catalog xe2x80x9cDIMACEL Compounds and Related Electrostatic Packagingxe2x80x9d.
Those skilled in the art could obviously think of numerous different applications for the invention. This will be the case for any detection of a predetermined substance or xe2x80x9canalytexe2x80x9d using a highly specific reaction with a corresponding xe2x80x9cligandxe2x80x9d. It will in all cases be possible, without the exercise of invention and if the need arises, to modify one of the constituents of the reaction to give it a polarity which will be neutralized, or even reversed in the final reaction product, wherein the method of the invention can also be applied to it. Similarly, those skilled in the art will in each case be able to adapt the method of the invention, for example to choose polarities to be given to the electrodes to render it operational for the detection of any analyte adapted to form a specific compound with a given ligand, when this formation is also accompanied by a neutralization, or even a reversal of the electric charge, either of the analyte, or of the ligand.
Thus more generally, when the polarities of the charges carried by the ligand and those of the electrodes are of opposite sign, the complex is detected in the liquid having migrated between the two electrodes when the initial having migrated between the two electrodes when the initial liquid contained the analyte.
Furthermore, when the polarities of the charges carried by the ligand and that of the electrodes are of the same sign and the polarities of the charges carried by the analyte and the complex itself are of opposite sign, there will be detected only the possible excess of ligand initially used when the analyte was initially present.
As has already been mentioned, it is advantageous that the ligand bear a marker, in particular a marker transformable into a colorometric, fluorescent or chemiluminescent signal and that the signal zone comprise a detector of the marker, in the signal zone that the liquid reaches that has migrated through the porous material, between the electrodes.
The signal zone is itself preferably formed in a porous material in contact with that which is interposed between the electrode, so as to permit access in the signal zone to the liquid having migrated first through the porous material interposed between the electrodes, and then through the porous material comprising the signal zone.
In one of its preferred embodiments, the method according to the invention thus uses the electrical attraction or repulsion exerted by two or more electrodes (preferably thin and flat), on one of the constituents of the analyte-ligand couple, for example on immunoglobulins (lg) accordingly as the immunoglobulins are bound or not to their specific antigen. A microdrop of specimen to be tested is disposed on the porous material, itself generally formed by a thin sheet, upstream of the latter or in the upstream region of the electrode pair, the drop of specimen being then absorbed immediately by the porous sheet, often an insulating paper. Then there is deposited a volume of marked antibody solution which will mix with the previously deposited drop of specimen. If the antigen that is sought is present in the drop of specimen, it will be rapidly captured by the marked antibodies. The electrodes will then differentiate between an antibody complexed with the antigen and an antibody which is not encountered antigen in the droplet, in particular when traversed by a negative electric current. In this case, the electrodes capture the NH2 sites (positively charged) of the immunoglobulins which are not bonded to an antigen, and permit migration of the immunoglobulins (lg) whose NH2 site is this time saturated and masked by an antigen in the porous sheet, between the two electrodes, up to the signal zone in which they can be detected and measured, when they have first been prepared before the determination (conjugated with an enzyme or a radioactive marker or any other means).
But these electrodes can also be positively charged or be traversed by a positive current, this time repelling the NH2 sites of the immunoglobulins (lg) that have remained free from any bonding to an antigen. They will on the other hand capture the lg+antigens complexes which all have a sufficient number of negative charges, thus captured by this positive electrode. There thus occurs an inversion of the signal at the end of preparation: the signal is supplied only by the lg remaining free from any antigen.
Combinations of pairs of electrodes can also be used, associated with the porous material, in particular when it is sandwiched successively between a first pair and then, downstream of the latter, with a second pair of electrodes, the electrodes of this second pair being also charged and of identical polarities to each other, but of a sign opposite that of the polarities of the electrodes of the preceding pair, the detection being then carried out upstream of the second pair of electrodes, after the liquid has migrated through the porous material first through the first, then through the second pair of electrodes. This procedure thus permits also semiquantitative dosage, to the extent that the first pair of electrodes can retain only a predetermined quantity as the case may be, either the marked ligand, or of the complex formed, the final detection thus indicating only the excess as the case may be, either of the complex formed, or of the analyte initially present in the fluid or liquid that is studied.
Preferably the first pair of electrodes is of a sign such that, and is calibrated so as to trap a predetermined maximum quantity of the analyte in the form of an analyte-ligand complex, and there is detected downstream of the second pair of electrodes the excess of analyte which the fluid or a liquid initially contained.
This thus permits practicing semiquantitative determinations: the negative electrodes capture all the free immunoglobulins and the positive electrodes, which are smaller, capture only a portion of the lg+antigen complexes, this portion of the complexes corresponding to the physiological quantity of antigen that is not to be exceeded. If there is excess of this antigen, the positive electrodes could not capture all the complexes formed and a signal would thus be given, indicating the excess of this antigen beyond the physiological quantities.
An important advantage of the invention is the rapidity of determination. Between deposit of the specimen of the marked antibody solution, and the appearance of the result, takes hardly more than 5 to 10 seconds. The reason is that the attraction or repulsion of the antibodies is instantaneous, whilst if this reaction were entirely immunological (attraction of the complex antibody+antigen by a second antibody preliminarily fixed for example), an incubation would be necessary. Thus, the immunological reaction takes place in the insulating or electrically inert porous sheet immediately after the deposit. This immunological reaction can be accelerated by a factor of 10,000 times by the addition of suitable reagents (such as polyethylene glycol 8000 for example). The reaction which then takes place between the electrodes is thus not immunological but rather electrical, and hence instantaneous, which gives to this invention an essential quality for emergency determinations (for example, myocardial enzymes during infarction, forecast of an imminent embolism by determining D-Dimere or coagulation factors after bone surgery or in phlebitis, etc . . . ).
It is important that the antibodies comprise before the reaction free NH2 groups. It is thus preferable that the antibodies remain monomers and that the marking takes place without the production of aggregates or polymers of the antibodies using the free NH2 groups. In case of the formation of polymers or antibody aggregates, the NH2 sites of the aggregated antibodies risk being masked, which will reduce correspondingly their capacity to be captured, particularly by the negative electrodes. The process could nevertheless be used, but with less sensitivity, to the extent the antibodies comprise a sufficient number of NH2 groups adapted to intervene in the antigen-antibody reaction or, in the absence of antigen, to be captured in a sufficiently energetic way.
The preceding situation must in a very preferred way be avoided. Moreover, the marked antibodies (or other marked ligands) must be exempt from any free marker, even traces thereof, above all if there is involved an enzyme adapted to modify a substrate (or group of molecules forming a substrate). During production, for example, of conjugated antibodies marked with peroxidase by known techniques, it will be generally required to proceed with a purification of the marked antibodies by several molecules of peroxidase by techniques also known, particularly by chromatography of the reaction product so as to retain only the peaks of elusion containing the marked antibodies by several molecules of peroxidase and to eliminate all the others, plus those containing molecules of free peroxidase.
The marking of the monomeric antibodies can be carried out according to a very high ratio of molecules of marking per molecule of antibody, which can reach a value of 5. Recourse could also be had to mixtures of monoclonal antibodies, all marked, but recognizing epitopes respectively distinct from the antigen to be detected, which will increase all the more the sensitivity of detection.
The extreme sensitivity of the process in a number of its applications is due to several reasons:
the materials used are all inert (porous material or electrodes) and do not fix any molecule of a protein as do the ELISA methods responsible for important background noise which prevents sensitive measurements. Foreign protein or foreign antibody can be fixed, which gives rise to the total absence of background noise.
the signal zone contains the most sensitive reagents and the most rapid to react with the marking of the antibodies arrived there
finally, the high sensitivity of the electrodes does not leave any of the xe2x80x9ctrappablexe2x80x9d antibodies free, but above all does not trap any xe2x80x9cnon-trappablexe2x80x9d antibody molecule adapted to give the signal. What can give the slightest signal can only correspond to a marked antibody and not to background noise.
In the present invention, it is not only a matter of separating or causing to migrate differentially the different molecular weights; it is moreover a matter of detecting or rejecting immunoglobulins according to whether they are bonded or not to their specific antigen. The molecular weight plays no role because the technique works perfectly as well with Fab fragments from IgG (the lightest) as with IgM supposed to be heavier.
In the present invention described here, the electrodes do not support any reaction. They play only, during the short instant in which a fluid migrates through the porous material, their electrical role of repulsion or attraction. This is the result of the migration of the fluids after passage between these electrodes, which supplies a signal, and not the electrodes.
As a modification, the ligandxe2x80x94or analytexe2x80x94is deposited dry on a predetermined region of the porous material, in a region essentially upstream of the electrodes, the fluid or liquid presumed to contain the analytexe2x80x94or the ligandxe2x80x94being introduced into this zone of the porous material.
For example, it is the analyte which is deposited in said predetermined region essentially in a region upstream of the electrodes. Preferably, it is also the analyte which is the carrier of the electrical charges, the fluid or liquid presumed to contain it being thus introduced into the region where the ligand is deposited; after possible in situ production of the complex, a separate dose of the analyte itself is reintroduced into the same region, the latter being this time marked, and the possible presence of the marked analyte is detected in the liquid having migrated between the electrodes.
The invention also relates to a device for the detection of an analyte adapted to be contained in a liquid, by reaction with a specific ligand of the analyte to form a complex, the analyte or the ligand or both being carriers of neutralized or even opposite electric charges in the complex when the latter is formed, this device comprising:
at least one pair of two electrodes provided with surfaces which face each other and are electrically charged, with identical polarities, or adapted to be thus charged;
a porous material, itself electrically inert, interposed between the surfaces and permitting the diffusion within it, between said electrodes, of the liquid adapted to be deposited in a free region of this porous material, essentially upstream of its path of migration through this porous material;
means in combination with this porous material permitting the detection in the liquid having migrated through the latter, between said electrodes, of those of these components, analyte, ligand or complex, which have not been detected by the electrodes.
Preferably, the electrodes are formed of sheets that are statically charged or adapted to be thus charged and the porous material is itself formed of a sheet sandwiched between the two electrodes, this porous sheet extending downstream of said electrodes relative to the direction of migration and comprising in the external portion of the detection means, the constituents that have migrated.
This device is preferably provided with means for detecting a marker carried by one of the reagents, for example the ligand.
The means associated with said material or electrically inert porous sheet, are themselves preferably constituted by a porous sheet in contact with the first and through which can diffuse the liquid having migrated through the first to come into contact with the indicator contained in this second sheet.
In the description which follows, reference will be made more particularly to the situations arising the most frequently, for which the method and device of the invention are called upon to apply, those of the detection of particular antigens by predetermined specific antibodies (or vice versa).